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J. Biol. Chem., Vol. 277, Issue 40, 37888-37895, October 4, 2002

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Purification and Structural Characterization of the Central Hydrophobic Domain of Oleosin^*

Ming Li^§, Denis J. Murphy^¶, Ka-Ho K. Lee, Reginald Wilson^**, Linda J. Smith^**, David C. Clark^**, and Jao-Yiu Sung

From the  Department of Medicine & Therapeutics, 9/F, Clinical Building, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong, ^¶ Cambridge Laboratory, John Innes Canter, Norwich NR4 7UH, United Kingdom, the  Department of Anatomy, Chinese University of Hong Kong, Shatin, Hong Kong, and the ^** Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom

The oil bodies of rapeseeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with abundant structural alkaline proteins termed oleosins and some other minor proteins. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain flanked by relatively polar C- and N-terminal domains. Although the hydrophilic N-terminal domain had been studied, the structural feature of the central hydrophobic domain remains unclear due to its high hydrophobicity. In the present study, we reported the generation, purification, and characterization of a 9-kDa central hydrophobic domain from rapeseed oleosin (19 kDa). The 9-kDa central hydrophobic domain was produced by selectively degrading the N and C termini with enzymes and then purifying the digest by SDS-PAGE and electroelution. We have also reconstituted the central domain into liposomes and synthetic oil bodies to determine the secondary structure of the domain using CD and Fourier transform infrared (FTIR) spectroscopy. The spectra obtained from CD and FTIR were analyzed with reference to structural information of the N-terminal domain and the full-length rapeseed oleosin. Both CD and FTIR analysis revealed that 50-63% of the domain was composed of -sheet structure. Detailed analysis of the FTIR spectra indicated that 80% of the -sheet structure, present in the central domain, was arranged in parallel to the intermolecular -sheet structure. Therefore, interactions between adjacent oleosin proteins would give rise to a stable -sheet structure that would extend around the surface of the seed oil bodies stabilizing them in emulsion systems. The strategies used in our present study are significant in that it could be generally used to study difficult proteins with different independent structural domains, especially with long hydrophobic domains.

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